Summary

The differential distribution of the plant signaling molecule auxin is
required for many aspects of plant development. Local auxin maxima and
gradients arise as a result of local auxin metabolism and, predominantly, from
directional cell-to-cell transport. In this primer, we discuss how the
coordinated activity of several auxin influx and efflux systems, which
transport auxin across the plasma membrane, mediates directional auxin flow.
This activity crucially contributes to the correct setting of developmental
cues in embryogenesis, organogenesis, vascular tissue formation and
directional growth in response to environmental stimuli.

Introduction

The plant hormone auxin (the predominant form of which is indole-3-acetic
acid; IAA) is a major coordinating signal in the regulation of plant
development. Many aspects of auxin action depend on its differential
distribution within plant tissues, where it forms local maxima or gradients
between cells. Besides local biosynthesis and the release of active forms from
inactive precursors, the major determinant of differential auxin distribution
is its directional transport between cells. This regulated polar auxin
transport (PAT) within plant tissues appears to be unique to auxin, as it has
not been detected for any other signaling molecule. Molecular biology and
genetics approaches in the model system Arabidopsis thaliana have
contributed fundamentally to our understanding of the mechanisms of auxin
transport. Currently, a large body of evidence supports the concept that
intercellular auxin movement depends on several auxin-transporting mechanisms,
which include both passive and active processes that transport auxin over long
and short distances. Of these, the major mechanism for controlling auxin
distribution during plant development appears to be the active directional
cell-to-cell movement of auxin that is mediated by plasma membrane-based
influx and efflux carriers (see Glossary,
Box 1). Here, we summarize the
present state of knowledge on how the various auxin transport mechanisms
cooperate during plant development to fine-tune auxin distribution. We
describe the basic pathways of auxin transport and discuss auxin transport
routes during diverse developmental processes, such as embryogenesis, root and
shoot organogenesis, vascular tissue formation and tropisms (see Glossary,
Box 1).

Auxin transport systems in plants

In plants, auxin is generally transported by two distinct pathways.
Throughout the plant, most IAA is probably transported away from the source
tissues (young leaves and flowers) by an unregulated bulk flow in the mature
phloem (see Glossary, Box 1).
In addition, a slower, regulated, carrier-mediated cell-to-cell directional
transport moves auxin in the vascular cambium from the shoot towards the root
apex (Goldsmith, 1977), and
also mediates short-range auxin movement in different tissues. These two
pathways seem to be connected at the level of phloem loading in leaves
(Marchant et al., 2002) and
phloem unloading in roots (Swarup et al.,
2001).

A series of classical physiological experiments
(Box 2) predicted the existence
of carrier-type auxin influx and efflux components that mediate PAT. The
asymmetric cellular localization of these transporters has been proposed to
determine the direction of auxin flow. During the past two decades, candidates
for auxin carrier proteins and for the relevant regulatory mechanisms have
been identified (Fig. 1).
Heterologous expression experiments in cultured plant cells, yeast,
Xenopus laevis oocytes and mammalian cells have demonstrated the
auxin-transporting capacity of these carrier proteins
(Vieten et al., 2007).
Expression and localization studies of auxin carrier proteins, as well as
specific defects in differential auxin distribution
(Box 3) in plants that lack the
function of these carriers, established that carrier-dependent PAT is
absolutely required for the generation and maintenance of local auxin maxima
and gradients.

Influx carriers

For auxin influx, the characterization of an agravitropic (see Glossary,
Box 1) auxin resistant
1 mutant (aux1) of Arabidopsis that shows resistance to
an exogenous synthetic auxin, 2,4-D, led to the identification of the
AUX1/LIKE AUX1 (AUX1/LAX) family of transmembrane proteins, which are similar
to amino acid permeases, a group of proton-gradient-driven transporters
(Bennett et al., 1996;
Swarup et al., 2008). To
date, four auxin influx carriers with specific functions have been described
in Arabidopsis, and the functions of some homologs in other plants
have also been studied (Table
1). Recently, AUX1 and LAX3 has been shown to mediate IAA uptake
when heterologously expressed in Xenopus oocytes
(Yang et al., 2006;
Swarup et al., 2008), which
provides biochemical evidence for their role as auxin influx carriers.

Efflux carriers

The investigation of several Arabidopsis mutants, namely of the
allelic root mutants agravitropic 1 (agr1), wavy roots
6 (wav6) (Bell and Maher,
1990; Okada and Shimura,
1990) and ethylene insensitive root 1 (eir1)
(Roman et al., 1995), and the
floral mutant pin-formed1 (pin1)
(Okada et al., 1991), resulted
in the identification of auxin efflux carrier candidates. The root
agravitropic phenotypes, as well as the pin1 phenotype with defects
in organ initiation and phyllotaxy (see Glossary,
Box 1), can be phenocopied by
the pharmacological inhibition of auxin efflux. Additionally, these mutants
display decreased PAT in shoots and roots. The corresponding PIN1
gene encodes a plant-specific protein with two transmembrane regions separated
by a hydrophilic loop (Gälweiler et
al., 1998). Concomitantly, the agr1, wav6 and
eir1 mutants have been shown to be allelic with a mutant that carries
a mutation in another PIN family member, PIN2. The AGR1, WAV6,
EIR1 and PIN2 genes encode a homologous protein designated PIN2
(Chen et al., 1998;
Luschnig et al., 1998;
Müller et al., 1998;
Utsuno et al., 1998). Until
now, eight members of the PIN protein family have been isolated in
Arabidopsis and are commonly referred to as PIN1 to PIN8
(Vieten et al., 2007;
Zažímalová et al.,
2007). A subgroup comprising PIN5, PIN6 and PIN8 has a reduced
middle hydrophilic loop and presumably regulates the auxin exchange between
the endoplasmic reticulum and the cytosol
(Mravec et al., 2009). The
PIN1, PIN2, PIN3, PIN4 and PIN7 proteins, by contrast, are localized at the
plasma membrane, where they act as auxin efflux carriers
(Mravec et al., 2008;
Petrášek et al.,
2006). PIN homologs in other plants have also been identified
(Zažímalová et al.,
2007), and some of them have been functionally characterized
(Table 1).

Box 1. Glossary

Acropetal transport:

Transport of various compounds (including auxin) towards the tip of a
particular organ (stem or root).

Agravitropic:

Having defects in response to gravity. This defect might be a result of
a specific mutation. Horizontally placed agravitropic roots are unable to grow
downwards, agravitropic stems are unable to grow upwards.

Anticlinal division:

Cell division in a layer of cells that occurs perpendicular to the
plane of the cell layer.

Apical:

`Upper' side of the cell, facing the shoot apical meristem. Auxin
influx and efflux carriers: Integral plasma membrane proteins that
transport auxin molecules into and out of the cell, respectively.

Basal:

`Lower' side of the cell, facing the root tip.

Basipetal transport:

Transport of various compounds (including auxin) from the tip towards
the basis of the particular organ (stem or root).

Columella:

Group of cells in the central root cap, which contain plastids with
starch; the site of root gravity perception.

Plant secondary polyphenolic metabolites. Besides playing a role in
defense responses to environmental impact, they have been shown to modulate
auxin transport by their preferential effect on ABCB auxin
transporters.

Hypophysis:

The most apical cell of the suspensor, which forms the attachment
between the suspensor and the developing embryo. It gives rise to the
embryonic root of a plant, the radicle, which develops into the primary
root.

Periclinal division:

Cell division in a layer of cells that occurs parallel to the plane of
the cell layer.

Phloem:

Part of the vasculature that transports metabolites from the source
tissues (leaves) to other tissues.

Phyllotaxy:

The typically regular arrangement of leaves or floral organs, which
initiates at the shoot apical meristem.

Primary root:

The first root that develops from the embryonic root of a plant embryo,
the radicle.

Stele:

The central part of the root or stem that contains the vascular
tissue.

Suspensor:

Single cell file formed from the zygote daughter basal cell by
transverse divisions. This cell file connects the embryo with mother tissues
and later degenerates.

Tropisms:

Directional plant growth responses to various environmental stimuli,
such as light (phototropism) or gravity (gravitropism). The response always
depends on the direction of the stimulus and could therefore be positive or
negative (towards or away from the stimulus, respectively).

Vasculature:

Complex conductive tissue that consists of specialized cells that
transport water and nutrients from roots (xylem), cells that transport
products of photosynthesis and other metabolites from source tissue (phloem)
and several other cell types that form supporting tissues. In both xylem and
phloem, various plant hormones have been detected.

A model for the mechanism that underlies the directionality of cell-to-cell
auxin transport was proposed simultaneously by Rubery and Sheldrake
(Rubery and Sheldrake, 1974)
and Raven (Raven, 1975), and
is known as the chemiosmotic polar diffusion model
(Goldsmith, 1977). According
to this model, an undissociated lipophilic form of the native auxin molecule
(IAA) can easily enter the cell cytoplasm from a slightly acidic extracellular
environment (pH 5.5) by passive diffusion. As the pH of the cytoplasm is more
alkaline (pH 7) than the extracellular environment, a difference that is
maintained by the plasma membrane-located H+-ATPase, more IAA
molecules dissociate after entering the cells, and the resulting hydrophilic
auxin anions (IAA-) are trapped in the cytosol. The exit of
IAA- was therefore proposed to be assisted by active auxin anion
efflux carriers that constitute the limiting step and the major control units
of auxin transport. The directionality of auxin transport was postulated to be
attributable to the asymmetric distribution of such carriers at a particular
side of the cell (Goldsmith,
1977), which would steer auxin flow in the direction of the
predominant localization of the transporters. Early experiments with
suspension-cultured crown gall cells of Parthenocissus tricuspidata
(Rubery and Sheldrake, 1974)
also suggested the existence of active auxin anion uptake carriers that
probably act as 2H+ cotransporters.

Other proteins that play a role in auxin efflux are plant orthologs of the
mammalian ATP-binding cassette subfamily B (ABCB)-type transporters of the
multidrug resistance/phosphoglycoprotein (ABCB/MDR/PGP) protein family
(Noh et al., 2001;
Verrier et al., 2008). Some
of these (ABCB1, ABCB4 and ABCB19) have been identified as proteins with
binding affinity to the auxin transport inhibitor 1-naphthylphthalamic acid
(NPA) (Murphy et al., 2002;
Noh et al., 2001). The
biochemical evidence for these ABCB proteins having a role in auxin transport
has been provided by heterologously expressing them in tobacco cells, HeLa
cells and yeast (Geisler et al.,
2005; Petrášek
et al., 2006; Santelia et al.,
2005; Terasaka et al.,
2005). The importance of the ABCB proteins for auxin
transport-related development has been also documented in other higher plants
(Table 1).

Recently, a system for comparative analyses of transport activities and the
structure of all three groups of auxin transporters (AUX1/LAX, PIN and ABCB)
has been established in Schizosaccharomyces pombe
(Yang and Murphy, 2009). It
represents a valuable tool for testing the cooperation between these
transporters, as well as with other regulatory proteins.

Other auxin transporter candidates exist, for example the members of a
group of aromatic and neutral amino acid transporters in Arabidopsis
(Chen et al., 2001) or the
transmembrane protein TM20 in maize (Zea mays)
(Jahrmann et al., 2005).
However, their contribution to the intercellular transport of auxin is still
unclear.

Auxin transport regulation

Various aspects of plant development are mediated by transport-dependent
differential auxin distribution within tissues. Conceptually, multiple signals
can be integrated to modulate auxin-dependent development, which highlights
the importance of regulating each auxin-transporting system individually.
Auxin itself seems to be one of the most important regulators of its own
transport. Earlier physiological observations on the role of auxin in the
formation and regeneration of vascular tissues led to the formulation of the
canalization hypothesis, which postulates that auxin acts to polarize its own
transport (Sachs, 1981). This
theory proposes that the initial diffusion of auxin away from a source
positively reinforces its own transport, which ultimately leads to the
distribution of auxin into narrow canals, and that this canalization is an
important part of the mechanism that underlies coordinated tissue
polarization.

Auxin transport across the plasma membrane and auxin-regulated gene
expression. (A) Schematic depiction of auxin transport across the
plasma membrane. Both passive diffusion and specific auxin influx and efflux
carriers are involved in the transport of auxin (IAA) across the plasma
membrane. Undissociated IAA molecules enter cells by passive diffusion (a),
whereas the less lipophilic, and therefore less permeable, dissociated auxin
anions (IAA-) are transported inside via auxin influx
2H+ cotransporters of the AUX1/LAX family (b). In the more basic
intracellular environment (c), IAA dissociates and requires active transport
through the PIN or ABCB efflux transporter proteins to exit the cell. Some
cytosolic IAA is transported by PIN5 into the lumen of the endoplasmic
reticulum (ER). This compartmentalization presumably serves to regulate auxin
metabolism (Mravec et al.,
2009). Whereas PIN transporter activity is supposed to use a
H+ gradient that is maintained by the action of the plasma membrane
H+-ATPase (d), and possibly also the vacuolar H+
pyrophosphatase (Li et al.,
2005), ABCB transporters have ATPase activity (e). (B)
Schematic depiction of auxin-regulated gene expression. Intracellular auxin
binds to its nuclear receptor from the TRANSPORT INHIBITOR RESPONSE 1/AUXIN
SIGNALING F-BOX (TIR1/AFB) family of F-box proteins, which are subunits of the
SCF E3-ligase protein complex (a). This leads to the ubiquitylation and the
proteasome-mediated specific degradation of auxin Aux/IAA transcriptional
repressors (b). Subsequently, the auxin response factors (ARFs) are
derepressed and activate auxin-inducible gene expression (c)
(Dharmasiri et al., 2005;
Kepinski and Leyser, 2005).
Among other auxin-responsive genes, all known auxin transporters are regulated
by this feedback mechanism (d). Ub, ubiquitin.

In general, the carrier-mediated transport of auxin can be regulated at
three levels: by the regulation of (1) the abundance of a carrier (by
regulating its transcription, translation and degradation); (2) subcellular
trafficking and targeting of auxin carriers to a specific position on the
plasma membrane; and (3) transport activity (e.g. through the
post-translational modification of carriers, the levels and activity of
endogenous inhibitors, the regulation of the plasma membrane pH gradient, the
composition of the plasma membrane and the interactions among individual
transporters or transport systems).

Transport can also be controlled by the incidence of transporters at the
plasma membrane (Box 4). This
mode of regulation has been demonstrated for some PIN proteins that undergo
constitutive internalization and recycling back to the cell surface
(Dhonukshe et al., 2007;
Geldner et al., 2001). It is
probably important for the establishment of
(Dhonukshe et al., 2008b), and
for dynamic changes in (Kleine-Vehn et
al., 2008a), PIN subcellular localization. Importantly, auxin
inhibits PIN internalization by an unknown mechanism, thus increasing the
amount and the activity of PIN proteins at the cell surface
(Paciorek et al., 2005). This
constitutes another, possibly non-transcriptional, mechanism for the feedback
regulation of auxin transport.

The regulation of PIN subcellular targeting is an effective way to modulate
auxin distribution because, consistent with classical predictions
(Box 2), the polar subcellular
localization of the PIN auxin efflux carriers has been shown to be important
for the directionality of auxin fluxes
(Wiśniewska et al.,
2006). Little is known about the mechanisms that control cell
polarity in plants; nonetheless, the phosphorylation of PIN is important for
decisions on PIN polar targeting. Analyses of Arabidopsis mutants
that have phenotypes typical for altered auxin transport, namely roots
curl in NPA 1 (rcn1) and pinoid (pid), have
led to the identification of the regulatory subunit of protein phosphatase 2A
(PP2A) (Deruére et al.,
1999) and the serine/threonine protein kinase PID
(Christensen et al., 2000) as
factors that are important for PIN targeting. The current model is that PID
phosphorylates PIN proteins, thus supporting their apical targeting, and that
PP2A antagonizes this action, thus promoting basal PIN delivery
(Friml et al., 2004;
Michniewicz et al., 2007).
Moreover, the Arabidopsis 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE
1 (PDK1) has been shown to stimulate the activity of PID kinase, which
provides evidence for a role of upstream phospholipid signaling in the control
of auxin transport (Zegzouti et al.,
2006). Similarly, the transcription factor INDEHISCENT (IND)
regulates PID expression, thus mediating auxin distribution-dependent fruit
development (Sorefan et al.,
2009). Conceptually, one can imagine that any signaling pathway
upstream of PID/PP2A has the capacity to modulate the transport-dependent
distribution of auxin by changing the balance between phosphorylation and
dephosphorylation. Interestingly, auxin itself regulates PID
expression (Benjamins et al.,
2001) and PIN polarity through TIR1-mediated signaling
(Sauer et al., 2006).

Despite the fact that auxin (indole-3-acetic acid; IAA) distribution plays
an important morphoregulatory role in plants, scientists still have no direct
method for tracking it in vivo at the cellular level and, instead, have to
rely on a set of more or less indirect techniques. For directly measuring the
endogenous IAA content, even in very small samples of plant tissue, gas
chromatography-mass spectrometry (GC-MS) is the most frequently employed
method (panel A) (Ljung et al.,
2005), but this technique lacks cellular resolution. To track
auxin distribution at the cellular level, antibodies against auxin carriers
(panel B) or IAA (panel C) are used
(Benková et al., 2003;
Friml et al., 2003a). However,
immunohistochemical staining procedures often suffer from technical problems
connected with the fixation of the rather diffusive IAA molecules, as well as
with the specificity of anti-IAA antibodies. Therefore, for noninvasive in
vivo tracking of auxin activity, synthetic promoters based on auxin-inducible
genes are employed (panel C) (Ulmasov et
al., 1997). These consist of multiple TGTCTC repeats of the
auxin-responsive element (designated DR5 or DR5rev in reverse orientation) and
can be coupled to markers, such as Escherichia coliβ
-D-glucuronidase (GUS) (Sabatini et
al., 1999), endoplasmic reticulum-localized Aequorea
victoria green fluorescent protein (GFP)
(Friml et al., 2003b), and a
nucleus-localized version of GFP or the modified yellow fluorescent protein
(YFP) version VENUS-N7 (Heisler et al.,
2005), to track their activity in plant tissues. Auxin-responsive
reporter constructs are widely used to get a preliminary impression of the
distribution of auxin activity, but their efficiency is limited by their
dependence on a comparable availability of the auxin signaling machinery in
all cells, nonlinear signal output, a relatively narrow concentration range
for detection, the time requirements of the transcription and protein folding
process, as well as the stability of the reporter molecules. For measurements
of auxin flow in plants, microscale assays with radiolabeled IAA have been
successfully adapted for Arabidopsis stem and root segments, and even
for whole seedlings (Lewis and Muday,
2009; Murphy et al.,
2000). More detailed information on the kinetic parameters of
auxin transporters can be obtained with the same technique in plant suspension
cultures (panel D) (Delbarre et al.,
1996; Petrášek
et al., 2006). An alternative, but yet not well established,
approach for measuring the actual flow of IAA at the tissue level utilizes
vibrating IAA-selective microelectrodes
(Mancuso et al., 2005), which
offer the advantage of noninvasive and continual recording of auxin flow.
Images are reproduced, with permission, from (A) Ljung et al.
(Ljung et al., 2001), (B)
Mravec et al. (Mravec et al.,
2008), (C) Benková et al.
(Benková et al., 2003)
and (D) Petrášek et al.
(Petrášek et al.,
2006).

The composition of the plasma membrane provides the appropriate environment
for protein-protein interactions and can thereby determine how effective the
auxin flux across the membrane will be. Indeed, the sterol composition of
membranes, which depends on the activity of the enzymes STEROL METHYL
TRANFERASE 1 (SMT1) and CYCLOPROPYL ISOMERASE 1 (CPI1) has been shown to be
crucial for the positioning of certain PIN proteins in the plasma membrane
(Men et al., 2008;
Willemsen et al., 2003).
Plasma membrane composition is also important for the localization of ABCB19,
which has been found to be present in the detergent-resistant microsomal
protein fractions of Arabidopsis seedling tissue lysates
(Titapiwatanakun et al.,
2009). Such sterol- and sphingolipid-rich plasma membrane
microdomains presumably constitute important specialized sites at which ABCB19
and PIN1 might interact physically
(Blakeslee et al., 2007).
Moreover, ABCB19 stabilizes PIN1 in these domains, and presumably influences
the rate of PIN1 endocytosis and thus its incidence at the plasma membrane
(Titapiwatanakun et al.,
2009) (Box 4).

The developmentally regulated formation of auxin gradients depends largely
on the fine-tuning of auxin flow polarity by means of the differential
subcellular trafficking and targeting of the AUX1/LAX, PIN and ABCB auxin
transporters. As all of these transporters are integral plasma membrane (PM)
proteins, they are distributed by the general mechanisms of vesicle
trafficking. All auxin transporters have been shown to be constitutively
recycled between the plasma membrane and endosomal compartments (shown in
blue). The endocytosis step of PIN1 and PIN2 recycling depends on clathrin
(Dhonukshe et al., 2007) and
on the sterol composition of the plasma membrane
(Men et al., 2008;
Willemsen et al., 2003),
which also influences AUX1 trafficking
(Kleine-Vehn et al., 2006). It
is also crucial for the interaction between ABCB19 and PIN1 proteins
(Titapiwatanakun et al.,
2009). This interaction seems to be important for the
stabilization of PIN1 at the plasma membrane sterol-rich microdomain (SRM),
with the subsequent enhancement of its auxin transporting activity. Compared
with ABCB1, ABCB19 is a rather stable plasma membrane protein, and its
trafficking requires the activity of the GNOM-LIKE 1 (GNL1) guanine nucleotide
exchange factor for ADP-ribosylation factors (ARF-GEF)
(Titapiwatanakun et al.,
2009). PIN1 targeting to the basal plasma membrane is regulated by
GNOM, another ARF-GEF (Geldner et al.,
2003), whereas one or more additional ARF-GEFs mediate PIN
targeting to the apical plasma membranes
(Kleine-Vehn et al., 2008a).
The apical localization of AUX1 is maintained by the activity of another
ARF-GEF and is assisted by the endoplasmic reticulum accessory protein AXR4
(Dharmasiri et al., 2006).
ARF-GEF-dependent endosomal sorting is also involved in the trafficking of
PIN2 to the lytic vacuolar pathway through the prevacuolar compartment (PVC),
from which PIN proteins might be retrieved again into the trans-Golgi network
through the assistance of the retromer complex subunits SORTING NEXIN 1 (SNX1)
and VACUOLAR PROTEIN SORTING 29 (VPS29)
(Jaillais et al., 2006;
Kleine-Vehn et al., 2008b).
The ubiquitylation of PIN2 potentially plays a role in the subcellular
trafficking of PIN2 and further regulates the amount of PIN2 at the plasma
membrane (Abas et al., 2006).
Additionally, PIN proteins are targets of phosphorylation by PINOID (PID)
kinase and of dephosphorylation by protein phosphatase 2A (PP2A)
(Michniewicz et al., 2007);
their phosphorylation state might be crucial for determining PIN recruitment
into the apical or basal targeting pathways.

Little is known about the mechanisms that might regulate the activity of
auxin transporters directly. It is possible that an additional phosphorylation
of PIN, distinct from PID-dependent action and mediated by D6 protein kinases,
controls PIN auxin efflux activity
(Zourelidou et al., 2009).
Alternatively, PIN auxin transport activity might be regulated by chemical
inhibitors. These exogenous compounds, which have been known for decades, have
been valuable tools in physiological studies on auxin transport and include a
well-known inhibitor of auxin efflux, NPA
(Rubery, 1990), as well as a
well-known inhibitor of auxin influx, 1-naphthoxyacetic acid (1-NOA)
(Parry et al., 2001). Detailed
knowledge about the mechanisms by which NPA and similar compounds inhibit
auxin efflux is still lacking. NPA has a high affinity for binding ABCB-type
auxin carriers, but low-affinity binding sites have also been found
(Murphy et al., 2002). This
low-affinity binding might be related to the more general inhibitory effects
of some efflux inhibitors on actin cytoskeleton dynamics and PIN trafficking
processes (Dhonukshe et al.,
2008a; Geldner et al.,
2001). A group of naturally occurring substances that might act
analogously to auxin transport inhibitors are the flavonoids (see Glossary,
Box 1), endogenous polyphenolic
compounds that modulate auxin transport and tropic responses
(Murphy et al., 2000;
Santelia et al., 2008). Both
NPA and flavonoids regulate the activity of ABCB1 and ABCB19
(Bailly et al., 2008;
Geisler et al., 2003;
Murphy et al., 2002;
Noh et al., 2001;
Rojas-Pierce et al., 2007),
possibly through influencing interaction with the peripheral plasma membrane
protein TWISTED DWARF 1 (TWD1) (Geisler et
al., 2003; Bailly et al.,
2008).

The above-mentioned examples only constitute glimpses into how the auxin
distribution network might be regulated at different levels. Nonetheless, they
demonstrate the potential for various internal and external signals to
influence the throughput and the direction of intercellular auxin fluxes, and
thus to regulate auxin-dependent development.

Auxin transport routes during embryogenesis

Auxin and auxin transport is already important at the earliest stages of
plant development. The analysis of Arabidopsis mutants, combined with
the visualization of the auxin response by means of auxin-inducible promoters,
demonstrated that differential auxin distribution mediates important steps
during embryogenesis, such as apical-basal axis specification and embryonic
leaf formation. The concerted action of PIN1, PIN4 and PIN7 efflux carriers
(Friml et al., 2002a;
Friml et al., 2003b) is
required for the differential auxin distribution in embryogenesis
(Fig. 2). Individual PIN
proteins act redundantly, given that single pin mutants can still complete
embryogenesis, whereas pin1 pin3 pin4 pin7 quadruple mutants are
strongly defective in the overall establishment of apical-basal polarity
(Benková et al., 2003;
Friml et al., 2003b). In
contrast to pin mutants, mutants in other auxin transport components, such as
the abcb and aux1/lax mutants, are not defective in
embryogenesis, which suggests a major role for PIN-dependent auxin transport
in patterning the embryo.

Auxin gradients and auxin transporters during embryogenesis.
Schematic depiction of the auxin distribution and the localization of auxin
transporters during early plant embryonic development. Auxin distribution
(depicted as a green gradient) has been inferred from DR5 activity and IAA
immunolocalization (Benková et al.,
2003; Friml et al.,
2002a; Friml et al.,
2003a). The localization of the efflux transporters PIN1, PIN4 and
PIN7, as well as that of ABCB1 and ABCB19, is based on immunolocalization
studies and on in vivo observations of green fluorescent protein (GFP)-tagged
proteins (Dhonukshe et al.,
2008b; Friml et al.,
2003b; Mravec et al.,
2008). Arrows indicate auxin flow mediated by a particular
transporter; dotted lines indicate the cell type-specific localization of
particular auxin transporters with no obvious polarity. PIN7, localized at the
apical sides of the suspensor cells (s), transports auxin towards the apical
cell (a) that forms the pro-embryo; there, PIN1, which is localized at all
inner cell sides, distributes auxin homogenously. ABCB1 and ABCB19 cooperate
during this initial stage and are localized apolarly in all cells or only in
the uppermost suspensor cell, respectively. The crucial moment in the setting
of the basal end of the apical-basal embryonic axis occurs during the early
globular stage, when PIN1 starts to be localized basally in the pro-embryonal
cells, and PIN7 is simultaneously shifted from the apical to the basal plasma
membrane of suspensor cells. These PIN polarity rearrangements reverse the
auxin flow downwards and, with the aid of PIN4, lead to auxin accumulation in
the forming hypophysis (h) (see Glossary,
Box 1). At this stage, ABCB19
helps to maintain the auxin distribution in the outer layers of the embryo. In
triangular- and heart-stage embryos, bilateral symmetry is established through
auxin maxima at the incipient cotyledon (c) primordia. These auxin maxima are
generated by PIN1 activity in the epidermis; in the inner cells of cotyledon
primordia, however, PIN1 mediates basipetal auxin transport towards the root
pole. SAM, future shoot apical meristem.

Soon after the first anticlinal division (see Glossary,
Box 1) of a fertilized zygote,
increased auxin accumulation can be detected in the apical cell by the
activity of the auxin-inducible element DR5 or by IAA immunolocalization
(Box 3). This differential
distribution results from the activity of PIN7 that is localized apically in
the adjacent suspensor cells. At this stage, PIN1 presumably mediates the
uniform distribution of auxin between cells of the forming pro-embryo
(Fig. 2,
Box 4). Both ABCB1 and ABCB19
contribute to auxin transport during the early stages of pro-embryo formation
(Mravec et al., 2008). ABCB1
is localized to all suspensor cells (see Glossary,
Box 1) and pro-embryonal cells,
and ABCB19 localization is restricted to the suspensor-forming cells. Both
proteins are localized without obvious polarity. Later, during the early
globular stage, PIN1 gradually relocalizes to the bottom plasma membranes of
the embryo cells that face the uppermost suspensor cell, the hypophysis
(Kleine-Vehn et al., 2008a).
Simultaneously, the polarity of PIN7 shifts from apical to basal in the
suspensor cells (Fig. 2,
Box 4). These coordinated PIN
polarity rearrangements, which are later also supported by the action of PIN4,
lead to an apical-to-basal flow of auxin and to auxin accumulation in the
hypophysis. At this stage, the auxin distribution and response are crucial for
the specification of the hypophysis as the precursor of the root meristem.
Accordingly, mutants of the auxin-binding F-box proteins TIR1 and AFB
(Dharmasiri et al., 2005), and
of the downstream transcriptional regulators MONOPTEROS (MP, also known as
ARF5) and BODENLOS (BDL, also known as IAA12)
(Hamann et al., 2002;
Hardtke and Berleth, 1998),
show pronounced defects in embryonic root formation.

Afterwards, during the development of the heart stage of the
Arabidopsis embryo, additional auxin maxima are formed at the
positions of the two initiating cotyledons (see Glossary,
Box 1), mainly through the
action of PIN1 (Benková et al.,
2003). At this stage, the ABCB19 expression pattern is largely
complementary to that of PIN1 and shows the highest expression in endodermal
and cortical tissues (Fig. 2).
The pin1 abcb1 abcb19 triple mutants, in contrast to the single
pin1 or double abcb1 abcb19 mutants, are severely defective
in establishing auxin maxima and show fused cotyledons, which hints at a
synergistic genetic interaction between PIN1 and ABCB proteins
(Mravec et al., 2008). These
results indicate a role for both the ABCB-mediated and PIN-dependent auxin
transport pathways in the generation of differential auxin distribution at
different stages of embryogenesis.

Auxin gradients and auxin transporters in root and shoot
morphogenesis. (A) Schematic overview of the directional flow of
auxin in the shoot and root of Arabidopsis thaliana. Auxin maxima in
shoot- and root-derived primordia and the root apex (green) are maintained by
auxin flow towards the root and shoot apices (solid arrows) and reverse flow
towards the root and shoot basis (dashed arrows). In the shoot and in
shoot-derived organs (leaf primordia P1 and P2), auxin is transported towards
the tip in the epidermal layers and refluxed back through inner tissues
(future vasculature). In the root and in root-derived organs (lateral root,
LR), auxin is transported towards the tip through the interior of the
primordium and refluxed back through the epidermis. (B,C) Auxin
transporters in the root tip (B) and developing lateral roots (C). (D)
Auxin transport in the shoot apical meristem (SAM) and during phyllotaxis. See
the main text for details on the role of each individual transporter. Auxin
distribution (depicted as a green gradient) has been inferred from DR5
activity and IAA immunolocalization. The localization of auxin transporters is
based on immunolocalization studies and on in vivo observations of GFP-tagged
proteins (Benková et al.,
2003; Blakeslee et al.,
2007; Friml et al.,
2002a,b;
Friml et al., 2003b;
Heisler et al., 2005;
Lewis et al., 2007;
Reinhardt et al., 2003;
Swarup et al., 2008;
Swarup et al., 2001;
Wu et al., 2007). Arrows
indicate auxin flow mediated by a particular transporter; dotted lines depict
the cell type-specific localization of particular auxin transporters with no
obvious polarity.

Auxin and postembryonic root and shoot development

Auxin plays an important role in the patterning of both shoot and root
apices, as well as in the initiation and the subsequent development of root
and shoot organs. Increased auxin levels at the incipient positions of the
primary root and the cotyledons (see Glossary,
Box 1) during embryogenesis are
reflected in postembryonic development. Auxin maxima always mark the positions
of organ initiation and, later, of the tips of developing organ primordia
(Benková et al., 2003).
Correspondingly, the local application and production of auxin triggers the
formation of leaves or flowers (Reinhardt
et al., 2000) and of lateral roots
(Dubrovsky et al., 2008).
Auxin fluxes and maxima in root- and shoot-derived organ primordia are similar
and can be described in terms of fountain and reverse fountain models,
respectively (Benková et al.,
2003) (Fig. 3A). In
general, all three auxin transport systems, using PIN, ABCB and AUX1/LAX
proteins, contribute to postembryogenic auxin transport, although the exact
contribution of each of these cooperating transport systems to total auxin
transport remains unresolved.

Auxin transport routes during root development

In the primary root, auxin is transported acropetally (see Glossary,
Box 1) towards the root tip by
a PIN-dependent route through the vascular parenchyma and through the phloem,
with subsequent AUX1-dependent unloading into protophloem cells
(Friml et al., 2002a;
Swarup et al., 2001). Auxin
flow towards the tip is maintained by the action of basally localized PIN1,
PIN3 and PIN7 in the stele (see Glossary,
Box 1)
(Blilou et al., 2005;
Friml et al., 2002a). In the
columella (see Glossary, Box
1), the action of PIN3 and PIN7 redirects auxin flow laterally to
the lateral root cap and the epidermis, where the apically localized PIN2
mediates the upward flow of auxin to the elongation zone
(Friml et al., 2003a;
Müller et al., 1998)
(Fig. 3B). The PIN2-based
epidermal auxin flow is further supported by the action of AUX1
(Swarup et al., 2001) and
ABCB4 (Terasaka et al., 2005;
Wu et al., 2007), whereas
PIN1, PIN3 and PIN7 recycle some auxin from the epidermis back to the
vasculature (Blilou et al.,
2005). The concerted action of the PIN auxin efflux carriers is
one of the major determinants of pattern formation in root tips
(Fig. 3B). By concentrating
auxin in the quiescent center, the columella initiates, whereas surrounding
stem cells (Sabatini et al.,
1999) restrict, the expression domain of the auxin-inducible
PLETHORA (PLT) transcription factors. PLTs are the master regulators of root
fate and, in turn, are required for PIN transcription
(Blilou et al., 2005). The
ABCB1 and ABCB19 auxin transporters seem to play a supportive role in
controlling how much auxin is available for each PIN-based transport flow.
ABCB1 is expressed in all root cells, except for the columella
(Mravec et al., 2008), whereas
ABCB19 expression is restricted to the endodermis and the pericycle,
which might help to separate the acropetal and basipetal auxin fluxes in the
stele and the epidermis, respectively
(Blakeslee et al., 2007;
Mravec et al., 2008;
Wu et al., 2007).

Auxin transport is also crucial for lateral root initiation and development
(Fig. 3C). In pericycle cells,
auxin maxima specify the founder cells for lateral root initiation
(Dubrovsky et al., 2008).
Subsequent rounds of coordinated divisions form the lateral root primordium,
from which the lateral root emerges later. Indeed, the functionally redundant
network of PIN efflux carriers facilitates the auxin transport that is needed
for the correct development of lateral root primordia
(Benková et al., 2003).
During the initiation phase, PIN1 is localized at the anticlinal membranes.
The switch of the pericycle cell division plane from anticlinal to periclinal
(see Glossary, Box 1) is
accompanied by the redistribution of PIN1 to the outer lateral plasma
membranes of inner cells (Benková et
al., 2003). This guanine nucleotide exchange factor for
ADP-ribosylation factors (ARF-GEF)-dependent, transcytosis-like PIN1 polarity
switch (Kleine-Vehn et al.,
2008a) mediates the auxin flow towards the primordium tip, where
an auxin maximum is formed. At later stages, the PIN2-mediated auxin transport
away from the tip through the outer layers is established.

AUX1 significantly contributes to lateral root formation, probably by
controlling the overall auxin levels in the root tip (by unloading auxin from
the phloem) and its availability in the region of lateral root initiation (by
basipetal transport from the tip)
(Marchant et al., 2002). An
interesting role is reserved for LAX3, which is induced in cells around the
developing primordium, where it establishes the auxin maxima needed for the
specific production of cell-wall-remodeling enzymes, which is necessary for
lateral root emergence (Swarup et al.,
2008). The ABCB1 and ABCB19 proteins are also expressed and
required for lateral root formation, as indicated by the defects in the
abcb and pin abcb mutants
(Mravec et al., 2008;
Petrášek et al.,
2006).

Auxin transport routes during shoot development

In the shoot apical meristem (SAM), the main source of auxin is unclear,
but auxin is probably partly supplied by the phloem (as in the case of roots)
and by young developing organs in the vicinity. Auxin fluxes are largely
reversed in shoots when compared with roots. Auxin arrives at the organ
initiation sites through the epidermis layer L1 and is canalized through the
interior of developing primordia into the basipetal stream of the main shoot
(Fig. 3D). This stream is
mostly maintained by the activities of PIN1, localized basally in xylem
parenchyma cells (Gälweiler et al.,
1998), and of ABCB19 (Noh et
al., 2001), which, together with ABCB1, helps to concentrate auxin
flux in the vascular parenchyma (Blakeslee
et al., 2007; Geisler et al.,
2005).

Shoot lateral organs (leaves and flowers) are generated from the SAM in a
highly periodic phyllotactic pattern. In Arabidopsis phyllotaxis, the
137° angle between developing primordia is marked by auxin maxima at the
position of incipient primordia
(Benková et al., 2003;
Heisler et al., 2005). This
highly organized auxin distribution is maintained by the cooperative action of
AUX1, LAX1, LAX2 and LAX3 (Bainbridge et
al., 2008), as well as that of PIN1. PIN1 polarities in the L1
layer, which also undergo complex rearrangements relative to auxin maxima,
appear to be responsible for generating the phyllotactic pattern of auxin
distribution, whereas auxin influx activities largely restrict auxin to the L1
layer (Reinhardt et al.,
2003). Not only the positioning, but also the development of shoot
lateral organs is regulated by auxin distribution, with the maximum
concentration at the primordium tip, where it is maintained mainly by the
activity of PIN1, which transports auxin through the epidermis towards the
tip. From there, a new basipetal, PIN1-dependent, transport route is gradually
established through the interior of the primordium. This marks future
developing vascular tissues that will connect new organs with the pre-existing
vascular network (Benková et al.,
2003; Heisler et al.,
2005). ABCB1 and ABCB19 also contribute to the establishment of
this auxin sink (Noh et al.,
2001) (Fig. 3D).
Observations regarding the localization of the components of different auxin
transport systems, combined with the defects in the corresponding mutants,
show that all the transport systems that depend on ABCB, AUX1/LAX and PIN
proteins are involved in shoot-derived organogenesis.

Auxin gradients and auxin transporters during gravitropic response.
(A) Positive root gravitropism. In starch-containing, gravity-sensing
root cap cells, PIN3 is relocalized from a symmetric distribution towards the
newly established bottom side after gravistimulation
(Friml et al., 2002b) (left).
The auxin that is redirected to the lower side of the root tip is further
transported to the elongation zone by epidermal PIN2/AUX1-mediated flow, where
it inhibits cell growth and causes the downward bending of the root
(Luschnig et al., 1998;
Müller et al., 1998;
Bennett et al., 1996;
Swarup et al., 2001;
Swarup et al., 2005) (right).
(B) Negative shoot gravitropism. Gravity is detected in
starch-containing endodermal cells, where PIN3 is supposed to redirect auxin
laterally to the vasculature (stele) (left). After gravistimulation (right),
PIN3 is presumably (in analogy to the situation in roots) relocated to the new
basal side of endodermal cells, and auxin flow is redirected to the outer cell
layers along the bottom side of the shoot, where auxin stimulates cell
elongation and the subsequent upward bending of the stem
(Friml et al., 2002b). Arrows
indicate auxin flow mediated by a particular transporter; dotted lines
indicate the cell type-specific localization of particular auxin transporters
with no obvious polarity; black arrows indicate the gravity vector (left) and
the direction of bending (right).

Auxin in vascular tissue development

As indicated already by the role of PIN1-dependent auxin flow in the
establishment of new vasculature from shoot-derived organs, auxin and auxin
transport are among the major determinants of the organized development of
vascular tissues, which serve as the main distribution route for water and
nutrients. Auxin seems to be a major positional signal for vascular tissue
formation, because local auxin applications to responsive tissues are
sufficient to trigger the de novo formation of vasculature
(Sachs, 1991). As stated
before, the canalization model of auxin flow predicts a feedback regulation of
the auxin transport rate and polarity by a localized auxin source. Such a
mechanism would be adequate to gradually generate more concentrated auxin
channels that would determine the position of the new vasculature and explain
the vasculature formation seen in leaves after wounding or in newly initiated
organs. Indeed, multiple feedback regulatory loops of PIN-dependent auxin
transport have been identified. Auxin modulates PIN transcription
(Vieten et al., 2005), PIN
incidence at the plasma membrane (Paciorek
et al., 2005) and also PIN polar localization
(Sauer et al., 2006). For
example, during the formation of vascular veins in leaves, PIN1 directs auxin
towards a convergence point in the leaf epidermis, from where veins are being
initiated and where PIN1 expression and polar localization mark the
position of all future veins (Scarpella et
al., 2006) (Fig.
4). Similarly, after wounding, PIN1 is repolarized, and a new
transport route is set up that determines the position of the regenerating
vasculature. Importantly, local auxin application is sufficient to induce
PIN1 expression, polarization and the subsequent establishment of
PIN1-based auxin channels, thus essentially specifying the future vasculature
(Sauer et al., 2006). These
observations provide strong support for the canalization hypothesis and
suggest that the auxin-dependent polarization of PIN1 is a key event in
vascular tissue formation during a variety of developmental processes.

The role of other auxin transport mechanisms in this process is unclear,
but they might have supporting functions. For example, AUX1 presumably
facilitates auxin loading into and out of the phloem component of the vascular
transport system (Marchant et al.,
2002) (Fig. 4).
ABCB19 is mostly localized in the vascular bundle sheet cells and potentially
prevents auxin leakage from the vascular flow
(Blakeslee et al., 2007).

Auxin routes in tropisms

The role of auxin and auxin transport in the directional growth responses
of plants to light (phototropism) and to gravity (gravitropism) played a major
role in the discovery of auxin and in the formulation of the concept of plant
hormones (Darwin, 1880). The
negative gravitropism of stems, the positive gravitropism of roots and the
positive phototropic curvature of stems are characterized by the uneven
distribution of auxin at the different sides of stimulated organs. This
differential auxin distribution activates asymmetric growth and subsequent
organ bending (Went, 1974) in
a context-specific manner: whereas higher intracellular auxin concentrations
trigger elongation in shoots, they are inhibitory in roots.

In shoots, gravity is detected in endodermal cells (starch sheath cells),
where PIN3 is localized at the inner plasma membrane. The corresponding
pin3 mutants are partially defective in hypocotyl gravitropism
(Friml et al., 2002b). It is
likely, but has not been conclusively demonstrated, that, similar to the root
gravitropic response, the PIN3 relocation to the bottom side of endodermis
cells triggers auxin accumulation in the lower side of the shoot, where the
auxin response promotes growth and upward bending
(Fig. 5B).

Conclusions

As discussed here, the polarized transport of auxin is crucial for plant
development. In addition to the passive diffusion of auxin molecules across
plasma membranes, three active and mutually cooperating auxin-transporting
systems have been described so far. Whereas the PIN auxin transporters are the
primary determinants of directionality, AUX1/LAX and ABCB proteins mainly
generate auxin sinks and control auxin levels in the auxin channels. The open
questions for future studies include the identification of the core action of
the different auxin transporters, how exactly auxin is transported across the
plasma membrane, how this process is regulated and how individual transporters
cooperate. Furthermore, the analysis of the regulatory sequences in promoters
of genes that code for auxin transporters, together with the study of
crosstalk with other plant hormones, will be crucial for understanding how
this system is controlled by other signaling pathways. The wealth of available
genetic tools will significantly contribute to answering these questions;
however, more biochemical and structural biology work will also be needed, in
particular to address the issues of the precise mechanism of auxin movement
across the plasma membrane.

Footnotes

We apologize to all authors whose work is not cited here owing to space
constraints. We thank Martine De Cock for help in preparing the manuscript.
This work was supported by the Grant Agency of the Academy of
Sciences of the Czech Republic (J.P. and J.F.) and by the
Odysseus program of the Research
Foundation-Flanders (J.F.).

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